The normal eucaryotic cell cycle typically comprises four main stages. The replication of DNA and the production of histones occurs during the S phase. The period of DNA synthesis is flanked by two "gap" periods, the G.sub.1 and G.sub.2 phases, during which pre- and post-replication DNA repair may occur, and during which the cell continues to produce the cellular macromolecules required for cell division. After the G.sub.2 phase, the cell will divide into two daughter cells during the M phase.
After irradiation, normal mammalian cells in S phase generally enter a period where DNA synthesis is arrested. The period of arrest provides a "checkpoint" in the cell cycle which allows time for the repair of DNA lesions, and thereby prevents replication of a faulty DNA template. Irradiation induced inhibition of DNA synthesis has been the subject of intensive research for many years (Hartwell and Kastan, 1994, Science 266:1821-1828). Recent results have reported a direct link between the ability of human cells to arrest in G.sub.1 phase following irradiation, and the status of the p53 tumor suppressor gene (Kasten et al., 1991 Cancer Res. 51:6304-6311; Kuerbitz et al., 1992, Pro. Natl. Acad. Sci. USA 89:7491-7495). In brief, these studies linked irradiation with increased levels of p53 protein. Further studies have demonstrated that inhibitors of protein kinase C (PKC) may prevent enhanced p53 expression after irradiation (Khanna & Lavin, 1993 Oncogene 8:3307-3312). These data cumulatively suggest that both PKC and p53 may play a role in irradiation-induced arrest of G.sub.1 -S transition.
2.1. Ataxia Telangiectasia
Ataxia telangiectasia (AT) is an autosomal recessive, multi-system disorder characterized by progressive neuromuscular and vascular degeneration. AT is transmitted at an estimated frequency of one per 40,000 live births. AT patients exhibit cerebellar ataxia; oculocutaneous telangiectases; and various immune defects including underdevelopment of the thymus leading to recurrent sinopulmonary infections. Chromosomal breakage and rearrangement are common in AT cells which are abnormally sensitive to ionizing radiation. Moreover, both homozygous recessive AT patients and heterozygous carriers are predisposed to malignancy.
Onset of AT generally occurs by 3 years of age, and the first symptom is usually truncal ataxia (Woods, C. G. and Taylor, A. M. R. (1992) Quart. J. Med. 82:169-179). Truncal ataxia, which precedes appendicular ataxia, is characterized by deep tendon reflexes which become diminished or absent by age 8. Over time, patients lose large-fiber sensation. By their twenties and early thirties, many AT patients develop progressive spinal muscular atrophy which mostly affects the hands and feet. Familial studies revealed that idiopathic scoliosis and vertebral anomalies occurred in excess in the relatives of AT patients.
Ataxia was reviewed, inter alia, in The Merck Manual of Diagnosis and Therapy, 16th Ed. (1992) Merck Research Laboratories, Rahway, N.J. See also Sedgwick and Boder (1991) in Handbook of Clinical Neurology: Hereditary Neuropathies and Spinocellular Atrophies Vol. 16(60) (P. J. Ninken et al., edu.), pp. 347-423, Elsevier Science, Amsterdam.
2.2. AT-Associated Chromosomal Correlations
In the last five years it has become apparent that the basic defect underlying AT effects one or more of the enzymes concerned with DNA processing. Kuhnlein and Paterson (1990, Carcinogenesis 11:117-121) reported a 5-6 fold increase in activity for uracil DNA glycosylase and DNAse III/IV and a 2-3 fold increase in apurinic/apyrimidinic DNA-binding protein. A tight chromosomal linkage is found between AT and THY1, a glycoprotein which is a major cell surface constituent of thymocytes and neurons. In addition, genes of the immunoglobulin super family, including CD3 and NCAM, are located near the AT region of chromosome 11.
Aurias and Dutrillaux (1986a, Hum. Genet. 72:25-26; 1986b, Hum. Genet. 72:210-214) reported that AT patients tend to have a high frequency of chromosomal breakage, not involving the AT locus itself, which leads to both translocations and inversions. The sites of breakage and rearrangements mostly involve those regions of chromosomes 2, 7, 14, and 22 where the immunoglobulin genes (IgK, IgH, and IgL) and T-cell receptor genes (TCR-.alpha., -.beta., and -.gamma.) are located. Ig clusters are known hot spots for breakage and rearrangements and are associated with several diseases. Previously Croce et al. (1985, Science 227:1044-1047) had suggested that the oncogene TCL1 which is located in the region of the chromosome 14 breakpoint may be activated by chromosome inversion or translocation, perhaps in juxtaposition with the TCR-.alpha. gene. Russo et al. (1989, Proc. Natl. Acad. Sci. 86: 602-606) presented further evidence of a cluster of breakpoints in the region of the putative oncogene TCL1 in AT patients with chronic lymphocytic leukemia.
Shaham and Becker (1981, Hum. Genet. 58:422-424) identified an AT clastogenic (chromosome breaking) factor in the plasma of AT patients and in the culture medium of AT skin fibroblasts. This small peptide has a molecular weight in the range of 500 to 1000. Although clastogenic activity could not be demonstrated using cell extracts, cultured AT fibroblasts are reported to show spontaneous chromosomal recombination rates 30 to 200 times higher than found in cultured normal fibroblasts. Increased recombination, translocations and other chromosomal aberrations in lymphocytes, monocytes and fibroblasts undoubtedly contribute to increased cancer risk.
Abnormal V(D)J recombination, joining V segments of the TCR-.gamma. with J segments of TCR-.beta. occurs in peripheral blood lymphocytes of AT patients at a frequency 50- to 100-fold higher than normal. This frequency is roughly the same as the increase in the risk for lymphoid malignancy in these individuals. In addition, the J-.alpha. sequence has been implicated in some T-cell translocations which remove chromosomal material between q12 and q32 of chromosome 11 (Russo et al. (1989) Proc. Natl. Acad. Sci. 86: 602-606).
All of these examples strongly imply that the immunodeficiencies associated with AT are due to the physical loss or functional inactivation of genetic material. This hypothesis is further substantiated by the fact that AT homozygotes commonly display a 5- to 14-fold increase in the frequency of oral exfoliated cell micronuclei. In AT, this easily scorable cytogenetic abnormality can be used as a diagnostic tool to identify AT heterozygotes who commonly display an intermediate frequency of such micronuclei (Rosin et al., 1989 Hum. Genet. 83:133-138).
Bigbee et al. (1989, Am. J. Hum. Genet. 44:402-408) demonstrated an increased frequency of somatic cell mutation in vivo in individuals with AT. The authors speculated that the predisposition to somatic cell mutation may be related to the increased susceptibility to cancer in AT homozygotes. Heterozygotes for the disease did not appear to have a significantly increased frequency of such mutations.
2.3. AT-Associated Sensitivity To Radiation/Cell Cycle
Clearly AT cells are deranged in a signal transduction pathway that controls cell cycle arrest following DNA damage. The product of the gene responsible for AT operates upstream of the p53 protein, which plays a role in the G.sub.1 -S checkpoint that delays the cell cycle in cells with damaged DNA. In normal cells, p53 levels increase 3- to 5-fold by a post-translational mechanism after .gamma.-irradiation; however, augmented p53 expression, and its subsequent induction of GADD45, does not occur in irradiated AT cells (Kastan et al. (1992) Cell 71:587-597). Another consistent feature of AT cells is that they do not temporarily arrest DNA synthesis in response to irradiation. In fact, radioresistant DNA synthesis is widely considered as the "molecular signature" of AT cells.
Checkpoints at both the G.sub.1 -S and the G.sub.2 -M transitions (Hartwell (1992) Cell 71:543-546) allow the cell to delay progress through the cell cycle. Checkpoints are thought to serve as surveillance mechanisms which detect DNA damage, and initiate the proper signal transduction pathways required to allow time for DNA repair processes to run their course before the cell proceeds to the next phase in the cell cycle.
Painter and Young (1982 Proc. Natl. Acad. Sci. 77:7315-7317) showed that the G.sub.1 -S checkpoint is inoperative in cells from AT patients. If the DNA is not repaired, abnormalities which could contribute to tumor development become permanent during the S phase. In fact, lymphoid, breast and other cancers are known to be increased in individuals heterozygous for germ line mutations of either p53 or the gene causing AT (Swift et al. 1991 New Eng. J. Med. 325:1831-1836; 1987, New Eng. J. Med. 316:1289-1294).
Shiloh et al. (1989, Hum. Genet. 84:15-18) presented evidence that the extent of chromatid damage induced in the G.sub.2 phase of the cell cycle by a moderate dose of x-rays is markedly higher in AT cells than in normal controls. These data correlate with the inability of some AT cells to carry out DNA synthesis during the S phase of the cell cycle (Mohamed et al. (1987) Biochem. Biophys. Res. Commun. 149:233-238). Because patients with AT are unusually sensitive to x-rays, treatment of malignancy with conventional dosages of radiation can be fatal.
2.4. AT-Associated Biochemistry
AT patients usually show an increase in serum alpha-fetoprotein. This is consistent with immaturity of the liver and suggests that a functional ATM protein is required for normal tissue differentiation. Patients also show a decrease in immunoglobulins although different patients may show different immunoglobulin (Ig)-A, -E, and -G2 levels, ranging from normal to completely absent. DNA topoisomerase II, an enzyme that introduces transient double-strand breaks, are also expressed at abnormal levels in many, but not all, AT cell lines. These variations appear to be correlated with various chromosomal rearrangements as discussed above.
In contrast, the severity of sinopulmonary infections such as staphylococcal pneumonia, chronic bronchitis, etc. do not necessarily correlate with AT-associated immunodeficiency and may be related to other genetic factors.
Endocrine abnormalities such as gonadal dysgenesis or atrophy and an unusual form of diabetes mellitus in which glucose tolerance is markedly decreased have been reported. Experiments examining insulin resistance suggest the presence of antireceptor immunoglobulins in the plasma of AT patients (Bar et al. (1978) New Eng. J. Med. 298:1164-1171). Mental retardation is sometimes associated with AT, and some older patients may suffer a severe loss of short-term memory (Gatti et al. (1991) Medicine 70:99-117).
2.5. AT-Associated Malignancy
Patients with AT have a strong predisposition to malignancy, particularly lymphomas and chronic lymphatic leukemia. About one-third of patients develop malignancies during their shortened, less than 50 year, life-span. In general, lymphomas in AT patients tend to be of B-cell origin, and leukemias of the T-cell type. Neoplastic cells are often of thymic origin, and Saxon et al. (1979) New Eng. J. Med. 300:700-704) have suggested that malignant transformation of uncommitted T-lymphocyte precursors capable of differentiation contribute to the chronic lymphatic leukemia often reported for AT patients. Solid tumors, including medulloblastomas and gliomas, occur at elevated rates in AT patients (Gatti et al. (1991) Medicine 70:99-117).
Heterozygotes are also said to be predisposed to lymphomas, with a relative risk of developing cancer compared to the normal population of about 3.7 (Swift et al. (1991) New Eng. J. Med. 325:1831-1836). Using documented cancer incidence (rather than cancer mortality) in persons heterozygous for AT, relative risk of cancer of all types was 3.8 for men and 3.5 for women. The relative risk for breast cancer, the cancer most clearly associated with AT, in carrier women was 5.1. In two independent studies, 8 to 18 percent of patients with breast cancer were confirmed to be AT heterozygotes (Swift et al. (1987) New Eng. J. Med. 316:1289-1294; Pippard et al. (1988) Cancer Res. 48:2929-2932). More recently, the frequency of the ATM gene was estimated to be 0.005, which translates into 1% of the population being ATM heterozygous carriers. This, combined with a revised relative risk of approximately 3.9 for female carriers would result in about 4% of all breast cancers being attributable to the heterozygous presence of the defective form of the ATM gene. Ford and Easton, 1995, Br. J. Cancer 72:805-812. The ATM gene is located on 11q22-23. Wooster et al. (1993, Hum. Genet. 92:91-94) typed 5 DNA markers in this chromosomal region in 16 breast cancer families. They found no evidence for linkage between familial breast cancer and these markers and concluded that the contribution of AT to familial breast cancer is likely to be minimal.
2.6. Genetic Complementation
As early as 1977, Paterson et al. (Research in Photobiology. Plenum, New York) suggested the existence of 2 distinct types of ataxia telangiectasia. By 1988, Jaspers et al. (Cytogenet. Cell Genet. 49:259-263) had used genetic complementation studies on fibroblasts to identify six different genetic complementation groups. Four of these, called AB, C, D, and E are clinically indistinguishable, present no group-specific patterns of clinical characteristics or ethnic origin, and display frequencies among AT patients of approximately 55%, 28%, 14%, and 3%, respectively. Hernandez et al. (1993, J. Med. Genet. 30:135-140) cited evidence for the existence of these four complementation groups: AB, C, D, and E on chromosome 11q. The group D defect was corrected by transfer of genetic material from region 11q22-q23 into an AT affected fibroblast cell line and group E cells have a deoxyribophosphodiesterase deficiency. The existence of different complementation groups presumably reflects alterations in distinct intragenic functional domains, given that the disease is caused by a single gene (ATM).
2.7 Beta Integrins
The integrin family comprises 14 alpha subunits and 8 beta subunits (Hynes, 1992, Cell 69:11-25). A functional structure consists of one alpha and one beta subunit which partially extrudes from the cell. The receptor is a dimer which connects the cytoskeleton with the extracellular matrix proteins.
One of the primary roles of the integrins is cell adhesion. In their connection with the proteins of the extracellular matrix, integrins are in close proximity to growth factors and they act as anchors for individual cells such as platelets and lymphocytes. Internally, they interact with talin molecules of the cytoskeleton and provide a more stable structural framework for tissues such as the skin, organs such as the liver, and the arteries and veins of the vascular system.
In their transmembrane role, the alpha and beta integrins appear to be bidirectional signaling proteins. They are among a select few molecules that propagate messages from the inside of the cell to the outside. Signaling function is explained or modeled via conformational changes, specifically interaction between the alpha and beta subunits, associated with signal transduction. As signal receptors, these molecules regulate intracellular pH, intracellular free calcium, tyrosine phosphorylation of proteins, and inositol lipid turnover.
Slight alterations, even point mutations, can be correlated with the loss of signaling. Lack of appropriate integrin signaling may be associated with the failure to halt the cell cycle for repair of chromosomal damage following chemical or physical disruption (such as ionizing radiation) and result in the higher cancer incidence seen in AT patients and carriers.
Integrins play a role in the immune response through activation of lymphocytes and the maturation of B-cells. It also appears that integrins may be downregulated or absent in AT cells. The relative dearth of integrins could explain the structural and functional immaturity of the liver and some of the immune and metastatic complications which are often associated with AT. Finally, when the secretion of integrins is blocked, cells undergo apoptosis. This apoptosis could affect fetal development and result in the non-Mendelian ratios seen in the inheritance of AT. In particular, it appears that a deficiency of integrin beta subunit 1 characterizes the major genetic form of AT, namely complementation group A.
2.8. Present Methods Of AT-Diagnosis
Early-onset ataxia with telangiectasias permits diagnosis of AT. Before the appearance of telangiectases, clinical diagnosis is problematic because cerebellar ataxia and oculomotor apraxia are also typical of X-linked Pelizaeus-Merzbacher disease and Joubert's syndrome. Elevated levels of alpha-fetoprotein and carcinoembryonic antigen are the most useful clinical markers (Gatti et al. (1991) Medicine 70:99-117). Dysgammaglobulinemia, decreased cellular immune responses, and peripheral lymphopenia are supportive findings but they may or may not be expressed in all AT patients.
Henderson et al. (1985, Lancet 11:1242) devised a rapid diagnostic method based on the hypersensitivity of AT lymphocytes by gamma irradiation. Similar studies have employed fibroblasts or chorionic villus sampling. Shiloh et al. (1989, Hum. Genet. 84:15-18) used the extent of X-ray damage to chromatids in the G2 phase of AT heterozygous cells as a test of heterozygosity.
Painter and Young (1980, Proc. Natl. Acad. Sci. 77:7315-7317), however, questioned the reliability of this approach on the basis that radiosensitivity of AT cells may be caused by their failure to delay DNA synthesis after radiation damage (see sensitivity to radiation/cell cycle above).
The exfoliated cell micronucleus test is performed on cells from either the oral cavity collected by swabbing the mucosa or the urinary bladder obtained by centrifugation of fresh urine specimens. Micronuclei are membrane-bound, Feulgen-positive, acentric fragments which result from fragmentation of chromosomes during division of epithelial cells. Both AT homozygotes and heterozygotes can be identified by this method (Rosin and Ochs 1986, Hum. Genet. 74:335-340, 1989 Hum. Genet. 83:133-138).
2.9. Identification of the Gene for AT
A breakthrough in the study of AT occurred recently with the identification of the gene underlying AT (Savitsky et al., 1995, Science 268:1749-1753). This gene, denoted ATM (ataxia-telangiectasia mutated), encodes a putative transcript of 12 kb specifying a protein homologous to the cytoplasmic signal transducer phosphatidylinositol 3-kinase (PI3K). Recent studies on ATM-related genes containing PI3K motifs have uncovered a family of genes, namely, S. cerevisiae TOR1, TOR2, TEL1 and S. pombe rad3, Drosophila mei-41, and the human gene encoding DNA-damage protein kinase catalytic subunit (DNA-PK.sub.cs) (Zakian, 1995, Cell 82:685-687), which are more similar to one another than to classical PI3K. Notably, the cellular phenotype conferred by these ATM-like genes suggest their products have partially overlapping functions in mediating various cell-cycle checkpoints. This is perhaps not surprising, given that, as noted earlier, the extent of cell cycle deregulation is widespread in AT cells after radiation exposure (Beamish and Lavin, 1994, Int. J. Radiat. Biol. 65:175-184).